The aim of this project is to determine the effects of tRNA gene arrangement on tRNA gene transcription. The tRNA5Asn gene family of Drosophila is of interest as a model tRNA gene family since four widely distributed genomic loci which contain tRNA5Asn genes have been identified and the DNA sequences of some have been determined. Members from each of the four loci will be isolated and characterized with respect to structure (sequence and arrangement) and in vitro transcriptional activity. This study will establish the gene organization of this family and will show whether there are common arrangements of these gene at the four loci. In vitro transcription using homologous and heterologous cell-free extracts of tRNA5Asn genes in single and clustered permutations will be performed. The assays of RNA synthesis and stable complex formation will give basic information on the functioning of tRNA genes in their genomic arrangements. The use of the stable complex transcription assay will show whether tRNA genes function as individual and discrete transcription units in the genome or whether 5' flanking and neighboring tRNA genes affect their ability to be transcribed.